Natural hirudin is a mixture of closely related polypeptides each containing approximately 64 to 66 amino acids and having a molecular weight of approximately 6900 daltons. At least 20 natural variants of hirudin have been identified. Scharf et al., FEBS Letters 255 pp. 105 to 110 (September 1989). It is produced by the European medicinal leech Hirudo medicinalis. It prevents blood from clotting by forming an inhibitory 1:1 molecular complex with activated thrombin (approximately K.sub.iApp =10.sup.-11 to 10.sup.-14 M). Hirudin forms a very tight complex with thrombin, wherein over 40% of the hirudin structure intimately contacts the thrombin molecule and covers both the fibrinogen recognition site of thrombin and the fibrinogen cleaving (active) site of thrombin. Twenty-seven of the sixty-five residues of hirudin have contacts less than 4.0 .ANG. with thrombin. This close fit prevents both the binding and cleavage of fibrinogen by thrombin.
It is impractical to prepare natural hirudin in quantities necessary for therapeutic use. At least three recombinant hirudins are now available identical to native variants, except the recombinants lack the sulphate residue on the tyrosine at position 63 found in native variants. These recombinant hirudins show pharmacological properties very similar to native hirudin (Markwardt, Sem. Thromb. Hemostas. 15 pp. 269 to 282 (1989)). European Patent Application 87402696.6 shows the amino acid sequence of hirudin variants 1, 2, and 3 (HV1, HV2 & HV3).
Three regions of the hirudin molecule are now believed to be essential to the thrombin-hirudin high affinity interaction based on X-ray crystallography (Rydel, et al., Science 249 (1990) pp. 277. to 280) and structure-activity studies (Krstenansky, et al., J. Med. Chem. 30 (1987) pp. 1688 to 1691). First, the three NH.sub.2 -terminal amino acid residues at positions 1, 2, and 3 of hirudin form a parallel beta strand with Ser.sup.214 to Glu.sup.217 of thrombin and participate in several dozen non-polar interactions with side chains of amino acids in and around the active site of thrombin. Second, the NH.sub.2 -terminal domain of hirudin from approximately Thr.sup.4 to Pro.sup.48 is a compactly folded structure composed of four loops stabilized by three disulfide bridges and antiparallel beta structures. The main function of this domain is to position and facilitate the interaction of hirudin's NH.sub.2 -terminal tripeptide at the thrombin active site. Third, the COOH-terminal tail of hirudin (Glu.sup.49 -Pro.sup.60) binds in the anion-binding exosite of thrombin and terminates in a hydrophobic helical turn defined by the sequence Glu.sup.61 -Leu.sup.64. The exosite constitutes the fibrinogen binding recognition site of thrombin and is dominated by numerous polar and non-polar interactions. The presence of hirudin in the exosite prevents fibrinogen from being recognized.
Most research on the hirudin molecule has focussed on (i) determining the roles of various regions of the molecule in its interaction with thrombin, and (ii) making modifications to the molecule to increase the binding affinity between hirudin and thrombin and thereby reduce the necessary dose in therapeutic applications. Some research has focussed on prolonging the activity or half-life of hirudin in vivo, and other research has been in the area of immobilizing hirudin on surfaces used in medical devices which come in contact with blood to provide a non-thrombogenic surface.
a. Modifications to Increase Binding Affinity or to Prolong In Vivo Half-Life of Hirudin
PCT Application WO 85/04418 discloses recombinant HV2 where Lys.sup.24, Asn.sup.33, Lys.sup.35, Gly.sup.36, Asn.sup.47, Glu.sup.49, and Asn.sup.53 are replaced by Gln, Asp, Glu, Lys, Lys, Gln, and Asp respectively.
European Patent Application No. 87402696.6 discloses recombinant variants 1, 2, and 3 where Tyr 63 or 64 is replaced by Glu or Asp and Lys 47 or Asn 47 is replaced by Arg or His.
European Patent Application No. 89400621.2 also discloses amino acid sequence modifications, including those at positions 1, 2, 33, 35, 36, 47, and 63, which increase the in vivo half life of the molecule, increase the specificity of the molecule's interaction with cell surface receptors and increase resistance to carboxypeptidase degradation. Arg is placed in the 33 position, Thr or Ser or Asp are placed at position 35, and Ser is placed at position 36.
European Patent Application No. 89810521.8 describes mutations at the 53, 57, 58, 61, 62, and 63 positions which, depending upon the analog selected, provide increased or decreased antitnroubogenic activity.
U.S. Pat. No. 4,179,337 discloses the attachment of mass-increasing molecules such as polyethylene glycol to proteins.
Lazar, et al. describe mutations at position 3 in recombinant hirudin variant 1 (rHV1) where antithrombin activity was increased by replacing Tyr with Phe or Trp, and markedly decreased by replacement with Thr (J. B. Lazar, R. C. Winant & P. H. Johnson. J. Biol. Chem. 266 pp. 685-688 (1991)).
Johnson, P. H. 1 et al. in "Biochemistry and Genetic Engineering of Hirudin", Seminars in Throbosis and Hemostasis, Vol. 15 No. 13 (1989) at pp. 309 describes hirudin fragments having antithrombogenic activity. These fragments correspond to residues 42 to 65 and 51 to 65.
European Patent Application No. 89810522.6 describes mutations at positions 1, 27, 36, 47, 48, 49, 51 and 52. The substitution at 36 is Lys, Arg, Asn, Val, Leu or Gln. The substitution 27 is Gln, Asn, Leu, Arg, or Val. The substitution at 49 is Asn or Met. The substitution at 47 is Arg, Asn, Val, or Leu.
European Patent Application No. 89810676.0 describes mutations at positions 1, 2, 27, 36, 47, 57, 58, 61, and 66 where the position 27 substitution is Gln, position 36 substitution is Gln and position 47 substitution is Arg.
The following references disclose modifications at the C-terminal and N-terminal ends of the hirudin molecule: European Patent No. 142860; U.S. Pat. No. 4,801,576; U.S. Pat. No. 4,745,177; U.S. Pat. No. 4,767,742, and European Application No. 86102462.8.
U.S. Pat. No. 4,791,100 discloses mutations of hirudin in positions corresponding to, inter alia, 35 and 36, where at 35 the substitution is Glu and at 36 the substitution is Lys. It also discloses analogs having a greater number of amino acids than native hirudin and others having fewer amino acids than native hirudin.
b. Immobilization of Hirudin on Surfaces
European Application No. 89311022.1, European Application No. 89307922.8 to Ito, and references cited therein disclose the attachment of hirudin to surfaces. The data disclosed in the Ito application shows substantial loss of antithrombogenic activity occurred when the molecule was immobilized on the surface.
c. Fragments
Various COOH-terminal polypeptide fragments of hirudin are known to bind to thrombin, thereby inhibiting the binding and cleavage of fibrinogen by thrombin. The minimum length polypeptide required to exert inhibitory activity has been reported as Phe.sup.56 -Gln.sup.65 (J. L. Yrstenansky, T. J. Owen, M. T.
Yates & S. J. T. Mao. J. Mec Chem 30 pp. 1688-1691 (1987)). Addition of amino acid residues to this polypeptide to increase its length and provide the amino acid sequences found in the several hirudin variants up to and including the complete sequences Glu.sup.49 -Gln.sup.65 augments the thrombin inhibitory activity of the fragments; and, the sequence may be extended to include Gly.sup.42 -Gln.sup.65 without compromising the efficacy of the inhibitor. Moreover, the deletion of Gln.sup.65 from the polypeptides of these series provides an analogous series of useful thrombin inhibitory peptides.
In a further extension of this reasoning, numerous synthetic variations of the polypeptide sequences found in the natural hirudin variants (peptidomimetic analogs of hirudin peptides) have been prepared and found to possess thrombin inhibitory activity. Notable among them are those described in European Patent Application No. 89302159.2 and others utilizing non-protein amino acids (European Patent Application No. 89122451.1).
Maraganore, J. M. at al. in an abstract presented at a symposium entitled "Biomedical Horizons of the Leech" on Oct. 24-28, 1990 disclose synthetic peptides which bind to both the anion binding exosite and the active site. The peptides are called "hirulogs" and consist of (i) an active-site specificity sequence, (ii) a poly-Gly linker, and (iii) an anion binding exosite recognition sequence.
There are many variations possible on this model of bivalent thrombin inhibitors and in general, bivalent protease inhibitors. For example see J. M. Maraganore, P. Bourdon, J. Jablonski, K. L. Ramachandran and J. W. Penton,II. Biochemistry 29 pp. 7095-7101 (1990); J. DiMaio, B. Gibbs, D. Munn, J. Lefebvre, F. Ni, and Y. Konishi. J. Biol. Chem. 265 pp. 21698-21703 (1990).
European Application No. 89302160.0 discloses peptides of about 8 to 26 amino acids having the biological activity of hirudin.
European Application No. 89302159.2 discloses cyclicized synthetic fragments of hirudin having antithrombogenic activity.
It is a primary object of this invention to provide antithrombogenic hirudin analogs having amino acids available for attachment of spacer molecules. The analogs can be bound to a surface via a spacer molecule rendering the surface nonthroenic. Alternatively, the analogs can be bound to a polymer via a spacer molecule to increase the analogs' in vivo half life. It is a further object of this invention to provide nonthroibogenic materials comprising such analogs attached to surfaces. It is a further object of this invention to provide analogs attached to polymers.